Initial COVID OSCTR sample RNAseq QC analysis QC
Miles Smith
19 March, 2021
Data processing of RNAseq data
Overview of sample characteristics:
## 342 samples selected for analysis## 395 files found## The following files do not have a matching metadata entry:## [1] "AA05141" "AA05181" "X500006_2" "X500016_2" "X500017_2" "X500082_2" "X500110_2" "X500126_2" "X500161_2" "X500224_2" "X500471_2" "X500500_2" "X500519_2" "X500523_2" "X500531_2" "X500533_2" "X500534_2" "X500535_2" "X510008_2" "X510010_2" "X533070_2" "X533071_2" "X533181_2" "X533182_2" "X541038_2" "X541183_2" "X541728_2" "X541980_2" "X550004_2" "X590013_2" "X590015_2" "X590048_2" "X590098_2" "X590113_2" "X500133_2" "X500544_2" "X500545_2" "X500576_2" "X500584_2" "X500586_2" "X500587_2" "X500588_2" "X500589_2" "X500590_2" "X510033_2" "X541051_2" "X541052_2" "X541091_2" "X541191_2" "X598101_2" "S00727_V07059_2" "S01248_V07399_2" "AA04447"## All expected files found.Sample QC filtering
Samples with a PC1 > 2 (the red lines) and/or PC2 zscore > 2.5 (the green lines) were dropped from analysis. Points in red represent samples that were dropped.
| sample_name | visit_ref | subject_ref | sex | ethnicity | age |
|---|---|---|---|---|---|
| AA05198 | PCV|500802|2020-10-20 | PCV|500802 | Female | [AI] | 43 |
| AA05201 | PCV|500808|2020-10-29 | PCV|500808 | Female | [EA] | 77 |
| AA05222 | PCV|500810|2020-12-09 | PCV|500810 | Female | [AI] | NA |
| AA05081 | ORDRCC|500643|2019-08-12 | ORDRCC|500643 | Female | [AI] | NA |
| AA05197 | ORDRCC|541174|2020-11-11 | ORDRCC|541174 | Female | [EA] | NA |
| X541052 | ORDRCC|541052|2019-01-24 | ORDRCC|541052 | Female | [EA] | 66 |
| AA02818 | MDFL|541038|2017-04-20 | MDFL|541038 | Female | [EA] | 32 |
| AA02821 | MDFL|500531|2017-06-27 | MDFL|500531 | Female | [AA] | 60 |
| AA02817 | MDFL|500519|2017-04-20 | MDFL|500519 | Female | [EA] | 33 |
| AA02819 | MDFL|500006|2017-05-01 | MDFL|500006 | Female | [AA] | 42 |
Overall correlation between samples
Data quality
MA-plot
This MA plot is of shrunken log2 fold changes (using the apeglm package), in which actual fold changes are adjusted to compensate for the high variablity of genes with few counts.
Dots in red indicate a significant expression difference between Control and SLE.
Dispersion estimates
Surrogate Variable Analysis
Surrogate variables are unknown, unmodeled, or latent sources of noise hidden in the data. The sva package was used to estimate the first three surrogate variables which were then introduced into the DESeq design formula to control for their effects.
K-means clustering optimization
To determine the optimal k, the gap statistic was calculated for k = (3,20) (k = 2 was ignored because it just returns the two disease clusters). A higher coefficient means better intracluster simliarity and intercluster differences.
Sample Sanity check
To determine if there is a gross mixup of samples, examine the expression of sex-specific transcripts to ensure that only female-subject samples express “XIST” and male-subject samples "DDX3Y:
There appears to be one sample that is lacking a label but appears to express high levels of the female markers XIST and ZFX as well as the male markers PCDH11Y and PRORY. There’s also a couple of other samples that may have a mix of male and female markers, but otherwise no signs of gross sample mixup.
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## setting value
## version R version 4.0.4 (2021-02-15)
## os Ubuntu 20.04 LTS
## system x86_64, linux-gnu
## ui X11
## language (EN)
## collate en_US.UTF-8
## ctype en_US.UTF-8
## tz Etc/UTC
## date 2021-03-19
##
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## survival 3.2-7 2020-09-28 [2] CRAN (R 4.0.4)
## sva * 3.38.0 2020-10-27 [1] Bioconductor
## svglite 2.0.0 2021-02-20 [1] RSPM (R 4.0.4)
## systemfonts 1.0.1 2021-02-09 [1] RSPM (R 4.0.4)
## tibble * 3.1.0 2021-02-25 [1] RSPM (R 4.0.3)
## tidygraph 1.2.0 2020-05-12 [1] RSPM (R 4.0.4)
## tidymodels * 0.1.2 2020-11-22 [1] RSPM (R 4.0.3)
## tidyr * 1.1.3 2021-03-03 [1] RSPM (R 4.0.4)
## tidyselect 1.1.0 2020-05-11 [1] RSPM (R 4.0.4)
## tidyverse * 1.3.0 2019-11-21 [1] RSPM (R 4.0.4)
## timeDate 3043.102 2018-02-21 [1] RSPM (R 4.0.3)
## tune * 0.1.3 2021-02-28 [1] RSPM (R 4.0.3)
## tweenr 1.0.1 2018-12-14 [1] RSPM (R 4.0.4)
## tximport * 1.18.0 2020-10-27 [1] RSPM (R 4.0.4)
## txtq 0.2.3 2020-06-23 [1] RSPM (R 4.0.4)
## utf8 1.2.1 2021-03-12 [1] RSPM (R 4.0.3)
## uuid 0.1-4 2020-02-26 [1] RSPM (R 4.0.3)
## uwot * 0.1.10 2020-12-15 [1] RSPM (R 4.0.4)
## vctrs 0.3.6 2020-12-17 [1] RSPM (R 4.0.3)
## viridis * 0.5.1 2018-03-29 [1] RSPM (R 4.0.3)
## viridisLite * 0.3.0 2018-02-01 [1] RSPM (R 4.0.4)
## webshot 0.5.2 2019-11-22 [1] RSPM (R 4.0.4)
## WGCNA * 1.70-3 2021-02-28 [1] RSPM (R 4.0.4)
## withr 2.4.1 2021-01-26 [1] RSPM (R 4.0.3)
## workflows * 0.2.2 2021-03-10 [1] RSPM (R 4.0.3)
## xfun 0.22 2021-03-11 [1] RSPM (R 4.0.3)
## XML 3.99-0.5 2020-07-23 [1] RSPM (R 4.0.4)
## xml2 1.3.2 2020-04-23 [1] RSPM (R 4.0.3)
## xtable 1.8-4 2019-04-21 [1] RSPM (R 4.0.4)
## XVector 0.30.0 2020-10-27 [1] RSPM (R 4.0.4)
## yaml 2.2.1 2020-02-01 [1] RSPM (R 4.0.3)
## yardstick * 0.0.7 2020-07-13 [1] RSPM (R 4.0.3)
## zip 2.1.1 2020-08-27 [1] RSPM (R 4.0.3)
## zlibbioc 1.36.0 2020-10-27 [1] RSPM (R 4.0.4)
##
## [1] /usr/local/lib/R/site-library
## [2] /usr/local/lib/R/library